Heart development and pathological remodeling are controlled by a network of transcription factors and non- coding RNAs that coordinate the expression of genes involved in cardiomyocyte proliferation, morphogenesis, protein synthesis, and contractility. In response to injury or hemodynamic stress, the adult myocardium undergoes compensatory hypertrophic growth that is characterized by an increase in cardiomyocyte cell size and reactivation of fetal cardiac genes. Sustained hypertrophy is a major risk factor for the development of systolic dysfunction and the progression to clinical heart disease. Identifying novel regulators of cardiac growth is vital to the development of therapeutics for the treatment of heart disease, which remains a leading cause of mortality in the United States. Serum response factor (SRF) is a widely-expressed transcription factor that regulates the expression of both muscle-specific and growth factor-inducible genes. In response to extracellular cues, SRF associates with diverse cell-type and signal-responsive transcriptional coactivators to switch between opposing mitogenic and myogenic gene programs that balance cardiomyocyte proliferation and differentiation. Myocardin is a potent coactivator of SRF that is essential for cardiac muscle differentiation and hypertrophy. We recently identified a novel long noncoding RNA (lncRNA) transcribed upstream of the myocardin locus, that we named the myocardin-associated long noncoding RNA, or CARDINAL. CARDINAL is significantly upregulated with myocardin during heart failure in both humans and mice, suggesting it plays an important role in the gene expression programs required to maintain normal heart function and ventricular remodeling in response to cardiac injury. In preliminary studies, we found that CARDINAL was robustly activated by myocardin and is a nuclear lncRNA that associates with chromatin. Genetic disruption of CARDINAL in mice resulted in ectopic expression of SRF-regulated mitogenic genes and decreased cardiac function. Interestingly, we found that CARDINAL forms a complex with SRF, suggesting it functions as the first described lncRNA coregulator of SRF-dependent gene networks in the heart. Long noncoding RNAs are an emerging class of transcriptional coregulators that remain largely unexplored, in part due to the difficulty in determining their binding partners and target genes. In this proposal, we will determine the role of CARDINAL in directly mediating cardiac gene expression using biochemical, cell- based, and unique animal models to investigate the molecular and biological significance of CARDINAL in the heart. We have developed a novel yeast three hybrid approach to identify protein binding partners for lncRNAs, which we will utilize to identify and characterize components of the CARDINAL-SRF regulatory complex. These studies will further our knowledge of the basic mechanisms controlling cardiac gene transcription, and will be broadly useful for investigating lncRNA-protein interactions in other systems. ! ! ! . !
The development and repair of the heart is governed by a network of protein-coding transcription factors and non-coding RNAs that coordinate the expression of genes involved in muscle cell proliferation, morphogenesis, differentiation, and contractility. This study aims to determine the molecular mechanisms underlying the function of a novel cardiac long noncoding RNA that functions as a transcriptional coregulator of human cardiac gene networks. These findings will provide novel insights into the pathways that govern cardiomyocyte differentiation, generate novel methodologies to uncover human lncRNA function and reveal new targets and strategies to treat cardiovascular disease.